Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape
Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are respo...
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Frontiers Media S.A.
2021-02-01
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Series: | Frontiers in Cellular and Infection Microbiology |
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Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2020.592022/full |
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Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yohan Ricci Zonta Ana Laura Ortega Dezen Amanda Manoel Della Coletta Kaio Shu Tsyr Yu Larissa Carvalho Leandro Alves dos Santos Igor de Carvalho Deprá Rachel M. Kratofil Rachel M. Kratofil Rachel M. Kratofil Michelle Elizabeth Willson Michelle Elizabeth Willson Michelle Elizabeth Willson Lori Zbytnuik Lori Zbytnuik Lori Zbytnuik Paul Kubes Paul Kubes Paul Kubes Valdecir Farias Ximenes Luciane Alarcão Dias-Melicio Luciane Alarcão Dias-Melicio Luciane Alarcão Dias-Melicio |
spellingShingle |
Yohan Ricci Zonta Ana Laura Ortega Dezen Amanda Manoel Della Coletta Kaio Shu Tsyr Yu Larissa Carvalho Leandro Alves dos Santos Igor de Carvalho Deprá Rachel M. Kratofil Rachel M. Kratofil Rachel M. Kratofil Michelle Elizabeth Willson Michelle Elizabeth Willson Michelle Elizabeth Willson Lori Zbytnuik Lori Zbytnuik Lori Zbytnuik Paul Kubes Paul Kubes Paul Kubes Valdecir Farias Ximenes Luciane Alarcão Dias-Melicio Luciane Alarcão Dias-Melicio Luciane Alarcão Dias-Melicio Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape Frontiers in Cellular and Infection Microbiology paracoccidioidomycosis neutrophils neutrophil extracellular traps (NETs) DNase escape mechanism |
author_facet |
Yohan Ricci Zonta Ana Laura Ortega Dezen Amanda Manoel Della Coletta Kaio Shu Tsyr Yu Larissa Carvalho Leandro Alves dos Santos Igor de Carvalho Deprá Rachel M. Kratofil Rachel M. Kratofil Rachel M. Kratofil Michelle Elizabeth Willson Michelle Elizabeth Willson Michelle Elizabeth Willson Lori Zbytnuik Lori Zbytnuik Lori Zbytnuik Paul Kubes Paul Kubes Paul Kubes Valdecir Farias Ximenes Luciane Alarcão Dias-Melicio Luciane Alarcão Dias-Melicio Luciane Alarcão Dias-Melicio |
author_sort |
Yohan Ricci Zonta |
title |
Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape |
title_short |
Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape |
title_full |
Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape |
title_fullStr |
Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape |
title_full_unstemmed |
Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape |
title_sort |
paracoccidioides brasiliensis releases a dnase-like protein that degrades nets and allows for fungal escape |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Cellular and Infection Microbiology |
issn |
2235-2988 |
publishDate |
2021-02-01 |
description |
Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action. |
topic |
paracoccidioidomycosis neutrophils neutrophil extracellular traps (NETs) DNase escape mechanism |
url |
https://www.frontiersin.org/articles/10.3389/fcimb.2020.592022/full |
work_keys_str_mv |
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doaj-62de1f1301e841b0b4526b7971f5e69d2021-02-10T08:50:48ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882021-02-011010.3389/fcimb.2020.592022592022Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal EscapeYohan Ricci Zonta0Ana Laura Ortega Dezen1Amanda Manoel Della Coletta2Kaio Shu Tsyr Yu3Larissa Carvalho4Leandro Alves dos Santos5Igor de Carvalho Deprá6Rachel M. Kratofil7Rachel M. Kratofil8Rachel M. Kratofil9Michelle Elizabeth Willson10Michelle Elizabeth Willson11Michelle Elizabeth Willson12Lori Zbytnuik13Lori Zbytnuik14Lori Zbytnuik15Paul Kubes16Paul Kubes17Paul Kubes18Valdecir Farias Ximenes19Luciane Alarcão Dias-Melicio20Luciane Alarcão Dias-Melicio21Luciane Alarcão Dias-Melicio22Laboratory of Immunopathology and Infectious Agents - LIAI, UNIPEX - Experimental Research Unity, Sector 5, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilLaboratory of Immunopathology and Infectious Agents - LIAI, UNIPEX - Experimental Research Unity, Sector 5, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilLaboratory of Immunopathology and Infectious Agents - LIAI, UNIPEX - Experimental Research Unity, Sector 5, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilLaboratory of Immunopathology and Infectious Agents - LIAI, UNIPEX - Experimental Research Unity, Sector 5, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilLaboratory of Immunopathology and Infectious Agents - LIAI, UNIPEX - Experimental Research Unity, Sector 5, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilConfocal Microscopy Laboratory, UNIPEX - Experimental Research Unity, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilLaboratory of Genetic Basis of Endocrinological Diseases, Experimental Research Unity (UNIPEX), Sector 5, São Paulo State University (UNESP), Botucatu, BrazilCalvin, Phoebe, and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaCalvin, Phoebe, and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaCalvin, Phoebe, and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaCalvin, Phoebe, and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaDepartment of Chemistry, Sciences School, São Paulo State University (UNESP), Bauru, BrazilLaboratory of Immunopathology and Infectious Agents - LIAI, UNIPEX - Experimental Research Unity, Sector 5, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilConfocal Microscopy Laboratory, UNIPEX - Experimental Research Unity, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilDepartment of Pathology, Medical School of Botucatu, São Paulo State University (UNESP), Botucatu, BrazilParacoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.https://www.frontiersin.org/articles/10.3389/fcimb.2020.592022/fullparacoccidioidomycosisneutrophilsneutrophil extracellular traps (NETs)DNaseescape mechanism |